The receptor tyrosine kinase ErbB-2 (also known as HER-2 or neu) is known to be amplified or overexpressed in approximately 30% of breast and ovarian caners. We previously have demonstrated that Akt is activated by ErbB-2 overexpression in breast cancer cells (Appendix 1). Recently, we have also shown that activation of Akt bound with and phosphorylated p21 Cip1/WAF1 and so resulted in the cytoplasmic localization of p21 Cip1/WAF1 and disrupted its cellcycle inhibitory activity (Appendix 2). Furthermore, activation of Akt can also induce the ubiquitination and degradation of p53 through Akt mediated MDM2 phosphorylation and thus confers cancer cells resistant to DNA damaging agents (Appendix 3). The regulations of p53-p21 cip1/WAF1 tumor suppressors are complicated as cytoplasm p21 Cip1/WAF1 has been speculated to possess anti-apoptotic function in addition to cell cycle inhibitor function which is known to occur in the nucleus. Furthermore, the p53 protein is controlled by post-translational modification through phosphorylation, acetylation, sumoylation, and ubiquitination, and thus controls its protein stability, cellular localization, DNA binding affinity and its interaction with other cellular proteins. The effect of Akt on the structural and functional relationship for p53 is largely unknown and has just initiated by the recent finding that Akt-mediated MDM2 phosphorylation enhances p53 degradation. Understanding the molecular mechanisms of regulation of p53-p21 Cip1/WAF1 by ErbB-2/Akt in breast cancer will not only increase our knowledge about the biology of cancer but also open a new avenue for rational drug designs for this deadly disease. In this application, wc will Cip1/ WAF1 further investigate the functional roles of cytoplasmic p21 Cip1/WAF1 (Specific Aim 1), the post-translational modification of p53 (Specific Aim 2), and physiological relevance of the phesphorylation of p21Cip1/WAF1 and MDM2 by PI-3K/Akt and their roles on the tumorigenesis of breast cancer will be examined in vivo using knock-in meuse models (Specific Aim 3). To achieve these goals, the three specific aims of this application are: Specific Aim 1: Molecular Mechanism of Anti-apoptotic function of cytoplasmic p21cip1/WAF1 Speeifie Aim 2: Molecular mechanism of Akt-mediated p53 funcitons Specific Aim 3: Functionality of cytoplasmic p21 cip1/WAF1 and phosphorylated MDM2